Microhomologies are prevalent at Cas9-induced larger deletions

Owens, Dominic; Caulder, Adam; Frontera, Vincent; Harman, Joe; Allan, Alasdair; Bucakci, Akin; Greder, Lucas; Codner, Gemma; Hublitz, Philip; McHugh, Peter J.; Teboul, Lydia; Bruijn, Marella F. T. R. de

doi:10.1093/nar/gkz459

Cited by 109 publications

(126 citation statements)

References 88 publications

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“…Our findings highlight the need for technologies that reliably detect all unwanted OnTE. Standard quality controls broadly performed in the field such as genotyping or karyotyping will only detect small events restricted to genotyping amplicons, or very large chromosomal aberrations, such as megabase-sized deletions, translocations and inversions, but miss the CRISPR-induced OnTE we and others revealed 5,7,8,11,15 . Moreover, high density SNP arrays faithfully detect only larger deletions, inversions and LOH, as their reliability increases with the number of affected SNPs.…”

Section: Discussionmentioning

confidence: 90%

“…However, application of CRISPR can be hampered by unwanted off-and on-target effects 3,4 . Recent studies in mice have described frequent occurrences of large deletions and complex rearrangements at CRISPR-edited loci after repair by non-homologous end joining (NHEJ) [5][6][7][8] . It is currently unclear if such alterations also affect clinically relevant human cells such as iPSCs, because repair pathways involved in CRISPR editing are differentially regulated 9 , as indicated for example by shorter human gene conversion tracts 2 .…”

Section: Introductionmentioning

confidence: 99%

“…Even though false identification of homozygously edited clones can corrupt the reliability of entire studies, tests for such deleterious OnTE are still lacking in the vast majority of genome editing studies. Some reports have applied primer-walk PCR [5][6][7][8] , PacBio or other deep sequencing methods [5][6][7] , or droplet digital PCR 7 to detect large on-target alterations, but these methods are expensive, laborious or require specific expertise and equipment. Here, we investigated whether large, mono-allelic deletions or insertions occur in human iPSCs after HDRmediated CRISPR genome editing and developed quantitative genotyping PCR (qgPCR) as a simple and universally applicable tool for their reliable identification.…”

Section: Introductionmentioning

confidence: 99%

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Detection of deleterious on-target effects after HDR-mediated CRISPR editing

Weisheit

Kroeger

Malik

et al. 2020

Preprint

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CRISPR genome editing is a promising tool for translational research but can cause undesired editing outcomes, both on-target at the edited locus and off-target at other genomic loci. We investigated the occurrence of deleterious on-target effects in human stem cells after insertion of disease-related mutations by homology-directed repair (HDR). We identified large, mono-allelic genomic deletions and loss-ofheterozygosity that escaped standard quality controls in up to 40% of edited clones. To reliably detect such events, we developed simple, low-cost and universally applicable quantitative genotyping PCR (qgPCR) as well as sequencing-based tools and suggest their usage as additional quality controls after editing. This will help to ensure the integrity of edited loci and increase the reliability of CRISPR editing.

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Section: Discussionmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Detection of deleterious on-target effects after HDR-mediated CRISPR editing

Weisheit

Kroeger

Malik

et al. 2020

Preprint

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“…Efficient exon deletion approaches implement CRISPR/Cas9 targeting of introns 5 and 3 of the target exon [38,39]. The distal DSB ends are subsequently repaired via classical EJ and alternative EJ repair pathways [15,25,73], frequently leading to highly efficient, homogenous indel outcomes. This is particularly true on the RNA level [39].…”

Section: Exon Deletionmentioning

confidence: 99%

Context-Dependent Strategies for Enhanced Genome Editing of Genodermatoses

March

Köcher

Koller

2020

Cells

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The skin provides direct protection to the human body from assault by the harsh external environment. The crucial function of this organ is significantly disrupted in genodermatoses patients. Genodermatoses comprise a heterogeneous group of largely monogenetic skin disorders, typically involving mutations in genes encoding structural proteins. Therapeutic options for this debilitating group of diseases, including epidermolysis bullosa, primarily consist of wound management. Genome editing approaches co-opt double-strand break repair pathways to introduce desired sequence alterations at specific loci. Rapid advances in genome editing technologies have the potential to propel novel genetic therapies into the clinic. However, the associated phenotypes of many mutations may be treated via several genome editing strategies. Therefore, for potential clinical applications, implementation of efficient approaches based upon mutation, gene and disease context is necessary. Here, we describe current genome editing approaches for the treatment of genodermatoses, along with a discussion of the optimal strategy for each genetic context, in order to achieve enhanced genome editing approaches.

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“…As such, the characterisation of mutant alleles in mosaic founders is particularly challenging. Events that are not captured by the chosen assays can be omitted (Shin et al, 2017;Kosicki et al, 2018;Owens et al, 2019) and screening can sometimes fail to distinguish rearranged from correct alleles in these complex animals (Codner et al, 2018). This suggests that the screen based on Sanger sequencing produced some false positives.…”

Section: Introductionmentioning

confidence: 99%

Application of long-read sequencing for robust identification of correct alleles in genome edited animals

Codner

Allan

et al. 2019

Preprint

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Recent developments in CRISPR/Cas9 genome editing tools have facilitated the introduction of more complex alleles, often spanning genetic intervals of several kilobases, directly into the embryo. These techniques often produce mosaic founder animals and the introduction of donor templates, via homologous directed repair, can be erroneous or incomplete. Newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the desired edit(s) together with founder mosaicism. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore long read sequencing of the targeted locus. Taking advantage of sequencing the entire length of the segment in each single read, we were able to determine whether the entire intended mutant sequence was present in both mosaic founders and their offspring.

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Microhomologies are prevalent at Cas9-induced larger deletions

Cited by 109 publications

References 88 publications

Detection of deleterious on-target effects after HDR-mediated CRISPR editing

Detection of deleterious on-target effects after HDR-mediated CRISPR editing

Context-Dependent Strategies for Enhanced Genome Editing of Genodermatoses

Application of long-read sequencing for robust identification of correct alleles in genome edited animals

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